Cell division is controlled by cyclin-dependent kinases (Cdks) which regulate cell cycle transitions. Acting in opposition to Cdks are the Wee1 kinases that phosphorylate and inactivate Cdks. Negative regulation imposed by Wee1 is relieved by the Cdc25 family of protein phosphatases, which dephosphorylate and activate Cdk2 and Cdc2. Cdks are a primary target of the DNA damage response and the absence of Cdk inhibition after DNA damage leads to radio-resistant DNA synthesis, a hallmark of the defects in the intra-S-phase checkpoint. Inactivation of Cdc25A in response to DNA damage is critical to the intra-S-phase checkpoint. We have determined the signal transduction pathway that leads to degradation of Cdc25A during both interphase and in response to DNA damage. Cdc25A degradation requires a multi-kinase cascade involving Chkl and an as yet unidentified kinase. Together, these kinases generate a phosphodegron on Cdc25A that is required for its rapid ubiquitination by the SCF-B-TRCP ubiquitin ligase and degradation by the proteasome. The identification of novel kinases involved in Cdc25A degradation and a determination of how they are regulated in response to DNA damage are central aims of this proposal. Cdc25A has also been suggested to be an oncogene and its levels are elevated in human cancer cells through both transcriptional and post-transcriptional mechanisms. However, the ability of deregulated Cdc25A to function as an oncogene has never been rigorously demonstrated. Both tissue culture based and in vivo experiments will be used to rigorously assess the ability of deregulated forms of Cdc25A to function as oncogenes. Although Cdc25A transcription is activated in a subset of cancers, relatively little is known concerning the mechanisms that regulate its expression. Positive and negative components in the Cdc25A regulatory pathway are possible points of deregulation in cancer. Genetic approaches in tissue culture cells will be used to identify proteins that positively and negatively regulate Cdc25A expression. Moreover, genome-wide shRNA screens will be used to identify new components of the DNA damage pathway using destruction of Cdc25 A as a marker for checkpoint function. In total, these experiments will define the mechanisms underlying Cdc25A regulation and will enhance our understanding of the involvement of Cdc25A deregulation in human cancer.